Acquisition of optimal TFH cell function is defined by specific molecular, positional, and TCR dynamic signatures

Abstract

The development of follicular helper CD4 T (TFH) cells is a dynamic process resulting in a heterogenous pool of TFH subsets. However, the cellular and molecular determinants of this heterogeneity and the possible mechanistic links between them is not clear. We found that human TFH differentiation is associated with significant changes in phenotypic, chemokine, functional, metabolic and transcriptional profile. Furthermore, this differentiation was associated with distinct positioning to follicular proliferating B cells. Single-cell T cell receptor (TCR) clonotype analysis indicated the transitioning toward PD-1hiCD57hi phenotype. Furthermore, the differentiation of TFH cells was associated with significant reduction in TCR level and drastic changes in immunological synapse formation. TFH synapse lacks a tight cSMAC (central supra molecular activation Cluster) but displays the TCR in peripheral microclusters, which are potentially advantageous in the ability of germinal center (GC) B cells to receive necessary help. Our data reveal significant aspects of human TFH heterogeneity and suggest that the PD-1hiCD57hi TFH cells, in particular, are endowed with distinctive programming and spatial positioning for optimal GC B cell help.

In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization

“…The adoption of laser-scanning confocal microscopy (LSCM) for image acquisition offers many advantages over traditional widefield methodologies including high lateral and axial spatial resolution as well as capacity for volumetric 3D analysis and high multiplexing (84), especially when iterative staining protocols, such as CyCIF are employed (85). Even though in this study we used tissues with a thickness of~10um, thicker tissues have successfully been imaged by us and others using immunofluorescence and confocal microscopy (24,86). To allow for increased accuracy in downstream image segmentation we imaged tissues at 40x magnification and recorded between 40000 and 70000 individual cells.…”

“…To allow for increased accuracy in downstream image segmentation we imaged tissues at 40x magnification and recorded between 40000 and 70000 individual cells. Furthermore, accurate representation and minimization of selection bias was ensured by imaging at least 50% of the tissue or by capturing 5 follicles on average in line with previously published research in our lab (24,30) We also opted to repeat selected markers among the different panels to address sampling errors arising from the analysis of a single tissue, measured populations with and without area normalization and examined key tonsillar B cell and Tfh cell populations by flow cytometry to further confirm the validity of our tissue analysis observations (Supplementary Figure 1A). Wherever possible, we used previously validated antibodies in our assays.…”

“…In line with the heterogeneity observed in other CD4 T-cell subsets, Tfh also display phenotypic heterogeneity that has been shown to associate with function (21,22). Furthermore, the exact topology of Tfh within GCs is also emerging as an important dimension for their analysis (23,24) HIV infection has been shown to perturb Tfh numbers both in humans as well as in NHP animal models of the disease (25,26). In addition, Tfh have been shown to represent an important reservoir of HIV during chronic infection (27,28).…”

“…Images were tiled and merged using LAS X Navigator software. To ensure accurate representation and minimize selection bias at least 50% of the tissue was imaged or 5 follicles were captured on average in line with previously published research in our lab (24,30). Replicate markers, shared among all panels, were also included in the analysis to ensure reproducibility across tissue sections and orientations.…”

“…Bcl-6 further distinguishes i) follicular B-cell subsets; GC B-cells (DZ: CD20 dim Ki67 + Bcl-6 + , LZ: CD20 hi/dim Ki67 +/-Bcl-6 + ) and non-GC (CD20 hi Ki67 -Bcl-6 -) and ii) Tfh cell subsets (PD-1 hi Bcl-6 hi/dim ) (Figure 1B). Furthermore, we used the marker CD57, an epitope expressed in a subset of CD4 T cells that are positive for CD69 and CD45RO and detected in approximately 15-25% of tonsil CXCR5+ CD4 T cells (33), to track two distinct GC-Tfh populations (CD57 + and CD57 -) that have been shown to possess divergent immunophenotypes (24). Potential regulatory CD4 T cells were also measured and localized based on the expression of the transcription factor FoxP3.…”